Methods for enhancing anti-tumor antibody therapy

ABSTRACT

Methods are provided to enhance the efficacy of antibody therapy directed to tumor cells.

CROSS REFERENCE

This application claims benefit and is a Continuation of applicationSer. No. 14/542,233 filed Nov. 14, 2014, which is a Continuation ofapplication Ser. No. 13/513,523 filed Aug. 21, 2012, now U.S. Pat. No.9,005,619 issued on Apr. 14, 2015, which is a 371 application and claimsthe benefit of PCT Application No. PCT/US2010/059221, filed Dec. 7,2010, which claims benefit of U.S. Provisional Patent Application No.61/358,303, filed Jun. 24, 2010, which claims benefit of U.S.Provisional Patent Application No. 61/287,067, filed Dec. 16, 2009,which claims benefit of U.S. Provisional Patent Application No.61/267,337, filed Dec. 7, 2009, which applications are incorporatedherein by reference in their entirety.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under contracts CA034233and CA153248 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

BACKGROUND

Monoclonal antibody technology is among the most notable scientificadvances in the last quarter century. Rapid translation of this researchhas prolonged the survival of thousands of patients with cancer. Thefirst approved monoclonal antibody, rituximab, a murine-human chimericIgG1 antibody against CD20, has become standard of care for patientswith B cell lymphomas. Monoclonal antibodies against HER2 (trastuzumab)and the EGF receptor (cetuximab) have similarly changed the naturalhistory of select patients with breast cancer, and both colorectal andhead and neck cancers, respectively.

Despite the promising activity of monoclonal antibodies, the responserates among patients with either refractory or advanced cancer aresuboptimal typically at less than 25%. Efforts to enhance the activityof monoclonal antibodies have focused on various combinations withcytotoxic chemotherapy. This largely ignores and may partiallyantagonize the immunologic mechanism by which monoclonal antibodiesfunction.

Both adaptive and innate immune cells participate in the surveillanceand the elimination of tumor cells. Among the innate cells are naturalkiller cells (NK cells), which constitute a major component of theinnate immune system. NK cells play a major role in the rejection oftumors and cells infected by viruses. The cells kill by releasing smallcytoplasmic granules of proteins called perforin and granzyme that causethe target cell to die by apoptosis.

Natural killer cell activity is tightly regulated, and requires anactivating signal. For example, activation of the Fc receptor byantibodies allows NK cells to lyse cells through antibody-dependentcellular cytotoxicity (ADCC). Upon activation, the NK cell releasesgranules containing granzymes and perforin. Perforin forms pores in thecell membrane of the target cell, through which the granzymes andassociated molecules can enter, inducing apoptosis.

ADCC is a primary mechanism by which tumor directed monoclonal antibodytherapy works. However, conventional cytotoxic chemotherapies inducemyelosuppression, decreasing the population of NK cells, therebyreducing the efficacy of ADCC. In contrast, therapies which augment NKcell function might offer the ability to improve activity of monoclonalantibodies without increasing toxicity to non-cancer cells. Clinicallythis is significant as an increasing population of cancer patientseither due to older age, advanced disease, or prior therapies, are notcandidates for conventional cytotoxic chemotherapy. The presentinvention addresses this issue.

SUMMARY OF THE INVENTION

Methods are provided to enhance the anti-tumor effect of monoclonalantibodies directed against tumor antigen(s). In the methods of theinvention, ADCC function is specifically augmented, which in turnenhances target cell killing, by sequential administration of anantibody directed against one or more tumor antigens, and an agonisticantibody against one or several inducible costimulatory molecules on NKcells.

An individual diagnosed with a tumor is first administered atumor-directed antibody. After a period of time NK cells which areinnate immune effector cells critical for ADCC upregulate expression ofinducible costimulatory molecules such as CD137, OX40, GITR, CD30 orICOS. Subsequently, a second antibody is administered targeting theinduced costimulatory molecule on NK cells (including but not limited toanti-CD137, anti-OX40, anti-GITR, anti-CD30 or anti-ICOS). In someembodiments, expression of the aforementioned costimulatory molecules isevaluated following administration of the tumor-directed antibody, inorder to determine the optimal time for dosing the second agent.Alternatively a timing period is determined empirically, and generallyapplied. The combination of agents and their sequential administrationis shown to provide for a level of tumor-specific, therapeutic synergythat is not observed with administration of the single agents alone. Themethod specifically enhances the anti-tumor function of monoclonalantibodies directed against tumor antigens. Because the second antibodytargets costimulatory molecules that have been inducibly expressed on NKcells by the tumor-directed antibody, this methods allows specificstimulation of NK cells which are implicated in ADCC-mediated killing ofthe tumor cells, while sparing other NK cells, thereby limitingpotential non specific side effects.

In some embodiments of the invention, the inducible costimulatorymolecule is CD137. In such methods, an individual diagnosed with a tumoris first administered a tumor-selective antibody. After a period of timesufficient to upregulate expression of CD137 in immune system cells, asecond agent is administered, which agent is an agonist of CD137. Insome embodiments the level of CD137 expression on blood cells isdetermined prior to each administering step, where an increase inexpression is indicative that the second agent may be administered.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures.

FIG. 1A-1C. Rituximab induces CD137 upregulation on human NK cellsfollowing incubation with CD20-positive tumor B cells. Peripheral bloodfrom three healthy donors was analyzed for CD137 expression on CD3⁻CD56⁺NK cells after 24 hour culture with lymphoma cell lines and trastuzumabor rituximab. (FIG. 1A) shows the percentage of CD137⁺ cells amongCD3⁻CD56⁺ NK cells from three healthy donors cultured with CD20⁻lymphoma cell line (OCI-Ly19) or CD20-positive lymphoma cell lines(Ramos, DHL-4, Raji) cell lines. (FIG. 1B) shows CD20 surface expressionon lymphoma cell lines (OCI-Ly19, Ramos, DHL-4 and Raji). Histogramswere colored according to the log 10-fold increase in WI of lymphomacell lines relative to isotype. (FIG. 1C) shows CD137 expression on NKcells subsets CD3⁻CD56^(bright) and CD3⁻CD56^(dim) from a representativehealthy donor after 24 hour culture with the CD20-positive lymphoma cellline, Ramos and rituximab.

FIG. 2A-2F. Anti-CD137 agonistic mAb increases rituximab-mediated NKcell cytotoxicity on tumor cells. NK cells isolated and purified fromthe peripheral blood of healthy donors were analyzed for degranulationby CD107a mobilization after 24 hour culture with five conditions: mediaalone; CD20-positive lymphoma cell line (Raji, Ramos, or DHL-4); tumorand rituximab; tumor and anti-CD137 antibody; or tumor, rituximab, andanti-CD137 agonistic antibody (FIG. 2A, Raji, *p=0.01; FIG. 2B, Ramos,*p=0.003; FIG. 2C, DHL-4, *p=0.002). NK cell cytotoxicity on Raji,Ramos, and DHL-4 tumor cells was analyzed in chromium release assay(FIG. 2D-2F). Preactivated NK cells (as described in Material andMethods) were purified before being incubated with chromium labeledRaji, Ramos, and DHL-4 cells for 4 hours. (FIG. 2D-2F) shows percentlysis of target cells by chromium release at varying effector (activatedNK cells):target (Raji) cell ratios cultured with media alone (♦),anti-CD137 (▴), rituximab (●), or rituximab and anti-CD137 (▪)antibodies (FIG. 2D, Raji, *p=0.01; FIG. 2E, Ramos, *p=0.01; FIG. 2F,DHL-4, *p=0.009).

FIG. 3A-3D. Anti-CD137 agonistic mAb enhances anti-lymphoma activity ofmurine anti-CD20 mAb in-vivo. C57BL/6 mice were inoculated with 5×10⁶BL3750 lymphoma tumor cells, subcutaneously, on the abdomen. FIG. 3A-3BPost-tumor inoculation, mice then received either Rat IgG control on day3 (●), anti-CD20 antibody on day 3 (▪), anti-CD137 antibody on day 4(♦), or anti-CD20 antibody on day 3 and anti-CD137 antibody on day 4(▴). Mice (10 per group) were then monitored for tumor growth (FIG. 3A,*p<0.001) and overall survival (FIG. 3B, *p=0.048). FIG. 3C-3D showtumor growth and survival with identical treatment sequence, howeverwith treatment delayed until day 8 post tumor inoculation. Mice receivedeither Rat IgG control on day 8 (●), anti-CD20 antibody on day 8 (▪),anti-CD137 antibody on day 9 (♦), or anti-CD20 antibody on day 8 andanti-CD137 antibody on day 9 (▴). Mice (10 per group) were thenmonitored for tumor growth (FIG. 3C, *p<0.001) and overall survival(FIG. 3D, *p<0.001).

FIG. 4A-4B. Anti-CD20 and anti-CD137 mAbs combination activity requiresappropriate sequence of mAb administration. C57BL/6 mice were inoculatedwith 5×10⁶ BL3750 lymphoma tumor cells, subcutaneously, on the abdomen.Post-tumor inoculation, mice then received either Rat IgG control on day3 (●), anti-CD20 mAb on day 3 and anti-CD137 mAb on day 4 (▴), oranti-CD20 mAb on day 3 and anti-CD137 mAb on day 3 (▪). Mice (10 pergroup) were then monitored for tumor growth (FIG. 4A, *p<0.001) andoverall survival (FIG. 4B, * p=0.001).

FIG. 5A-5D. Enhancement of the anti-lymphoma activity of anti-CD20 mAbby anti-CD137 agonistic mAb is dependent on NK cells and macrophages.(FIG. 5A) Peripheral blood cell subsets from lymphoma-bearing C57BL/6mice 4 days post-tumor inoculation treated on day 3 with either IgGcontrol or anti-CD20 antibody were analyzed for CD137 expression onCD3-NK1.1⁺ NK cells (NK), F4/80⁺ macrophages (Mφ), CD3⁺CD8⁺ T cells(CD8), and CD3⁺CD4⁺ T cells (CD4) (n=3 mice per group, *p=0.001). (FIG.5B) Tumor-infiltrating lymphocytes from lymphoma-bearing C57BL/6 mice 7days post-tumor inoculation treated on day 3 with either IgG control oranti-CD20 antibody were analyzed for CD137 expression on CD3-NK1.1⁺ NKcells (NK), F4/80⁺ macrophages (Mφ), CD3⁺CD8⁺ T cells (CD8), andCD3⁺CD4⁺ T cells (CD4) (n=3 mice per group, *p=0.012, NS=notsignificant). (FIG. 5C-5D) C57BL/6 mice were inoculated with 5×10⁶BL3750 lymphoma tumor cells. Post-tumor inoculation mice received eitherRat IgG control on day 3 (●), anti-Asialo-GM1 on days −1, 0, 5, 10, 15,20, and 25 with anti-CD20 antibody on day 3 and anti-CD137 antibody onday 4 (▪), liposomal (L.) clodronate on days −2, 0, 4, 8, 12, 16, 20,and 24 with anti-CD20 antibody on day 3 and anti-CD137 antibody on day 4(▾), anti-CD8 mAb on days −1, 0, 5, 10, 15, 20, and 25 with anti-CD20antibody on day 3 and anti-CD137 antibody on day 4 (♦), or anti-CD20antibody on day 3 and anti-CD137 antibody on day 4 (▴). Mice (10 pergroup) were then monitored for tumor growth (FIG. 5C, *p=0.002) andoverall survival (FIG. 5D, *p<0.001).

FIG. 6A-6C. Anti-CD137 agonistic mAb enhances anti-lymphoma activity ofrituximab in vivo in a disseminated human lymphoma xenotransplant model.SCID mice were inoculated with 3×10⁶ luciferase-labeled Raji lymphomatumor cells, intravenously through the retro-orbital sinus. Post-tumorinoculation, mice then received either Rat IgG control on day 3 (●),rituximab on day 3 (▪), anti-CD137 antibody on day 4 (♦), or rituximabon day 3 and anti-CD137 antibody on day 4 (▴). Treatment was continuedweekly for a total of 4 weeks. Luciferase imaging of representative mice10, 20, and 30 days post treatment are shown (FIG. 6A). Mice (5 pergroup) were then monitored for quantified bioluminescence (FIG. 6B,*p=0.001) and overall survival (FIG. 6C, *p=0.013).

FIG. 7A-7C. Rituximab-coated, autologous lymphoma cells induce CD137upregulation on NK cells from human patients with B cell malignancies.Peripheral blood from patients with B cell malignancies and circulatingtumor cells (CTC) were analyzed for CD137 expression on CD3⁻CD56⁺ NKcells after 24 hour culture with media alone, trastuzumab, or rituximab(FIGS. 7A and 7B). (FIG. 7A) shows CD16 and CD137 expression onCD3⁻CD56⁺ NK cells for a patient with marginal zone lymphoma (MZL) with70% CTC. (FIG. 7B) shows the percentage of CD137⁺ cells among CD3⁻CD56⁺NK cells in a cohort of 25 patients with follicular lymphoma (FL),chronic lymphocytic leukemia (CLL), MZL, mantle cell lymphoma (MCL),diffuse large B-cell lymphoma (DLBCL), and CD20-positive acutelymphoblastic leukemia (ALL). (FIG. 7C) shows correlation, R2=0.87,p<0.001, between the percentage of peripheral blood CTC and CD137surface expression on CD3⁻CD56⁺ NK cells after 24 hour culture withrituximab from patient samples with FL (

), CLL (●), MZL (

), DLBCL (

), and CD20-positive ALL (

).

FIG. 8A-8C. CD137 induction and temporal expression on NK cellsfollowing preactivation. Purified NK cells from healthy donors wereanalyzed for CD137 expression after 24 hour culture with media,rituximab, trastuzumab, lymphoma cell lines (Raji, Ramos, DHL-4, orOCI-Ly19) and rituximab (FIG. 8A). Purified NK cells from a healthydonor were analyzed for CD137 expression after 0, 4, 16, 24, 48 and 72hour culture with Raji cell line and rituximab (FIG. 8B). Forexperiments shown in FIG. 8C, peripheral blood mononuclear cells from arepresentative healthy donor were incubated with Raji, Ramos or DHL-4and rituximab for 24 hours. Preactivated NK cells were then analyzed forCD137 expression (FIG. 8C) prior to performing the cytotoxicity assay.

FIG. 9A-9B. Anti-CD137 agonistic mAb increases cytokine release andrituximab-mediated cytotoxicity of pre-activated NK cells. To evaluateNK cell interferon-γ secretion purified NK cells were isolated fromhealthy PBMCs and cultured for 24 hours together with rituximab (10μg/mL) and irradiated (5,000 rads) lymphoma tumor cells (Raji) at aratio of 1:1. After 24 hours, NK cells were isolated and assessed forpurity (>90% purity as defined by CD3-CD56+ flow cytometry)(FIG. 9A-9B).Preactivated, purified NK cells were then cultured for 4 hours in mediaalone, or with anti-CD137 mAb (BMS-663513, 10 μg/mL) alone, rituximab(10 μg/mL) alone, or rituximab plus anti-CD137 mAbs (both at 10 μg/mL)and supernatant was harvested and analyzed by ELISA for interferon-γ(FIG. 9A, *p=0.027). NK cell cytotoxicity on Raji tumor cells wasanalyzed in chromium release assay with and without prior NK cellpreactivation (FIG. 9B). Preactivated, and non-preactivated, purified NKcells were incubated with chromium-labeled Raji for 4 hours. Percentlysis of target cells by chromium release at varying effector(preactivated NK cells depicted in continuous line, and non-preactivatedNK cells depicted in dashed line):target (Raji) cell ratios culturedwith media alone (♦), anti-CD137 (▴), rituximab (●), or rituximab andanti-CD137 (▪) antibodies (*p=0.024).

FIG. 10. Anti-CD137 agonistic mAb increases rituximab-mediated NK celldegranulation. NK cells isolated and purified from the peripheral bloodof healthy donors were analyzed for degranulation by CD107a mobilizationafter 24 hour culture with media alone, CD20-positive lymphoma cell line(Raji, Ramos, or DHL-4), tumor and rituximab, tumor and anti-CD137antibody, or tumor, rituximab, and anti-CD137 agonistic antibody.Representative flow cytometry plot of CD107a expression on NK cellsafter culture with Ramos.

FIG. 11A-11C. Trastuzumab induces CD137 upregulation on human NK cellsfollowing incubation with HER2-positive tumor cells. Peripheral bloodfrom three healthy donors was analyzed for CD137 expression on CD3⁻CD56⁺NK cells after 24 hour culture with breast cancer cell lines and IgGcontrol, trastuzumab or rituximab. (FIG. 11A) shows the expression ofCD69⁺ and CD137⁺ on CD3⁻CD56⁺ NK cells from a representative healthydonor after 24 hour culture with HER2′ breast cancer cell line(BT474M1). (FIG. 11B) shows HER2 surface expression on breast cancercell lines (MCF7, BT474M1, SKBR3, and HER18). Histograms were coloredaccording to the log 10-fold increase in MFI of breast cancer cell linesrelative to isotype. (FIG. 11C) shows CD137 expression from threehealthy donors cultured on NK cells CD3⁻CD56⁺ after 24 hour culture withvariably expressing HER2 breast cancer cell lines (MCF7, BT474M1, SKBR3,HER18).

FIG. 12A-12C. Anti-CD137 agonistic mAb increases trastuzumab-mediated NKcell cytotoxicity on tumor cells as assayed by cell viability.Preactivated NK cells were purified before being incubated with MCF7,BT474M1, and HER18 for 18 hours. (FIG. 12A-12C) shows percent ofapoptotic target cells by annexin and 7AAD viability staining afterculture with NK cells, tumor, media alone, anti-CD137, trastuzumab, ortrastuzumab and anti-CD137 antibodies (FIG. 12A, MCF7 tumor line, FIG.12B, BT474M1 tumor line *p=0.03; FIG. 12C, HER18, **p<0.01).

FIG. 13. Anti-CD137 agonistic mAb increases trastuzumab-mediated NK cellcytotoxicity on tumor cells as assayed by chromium release. NK cellcytotoxicity on BT474M1 tumor cells was analyzed in chromium releaseassay. Preactivated NK cells were purified before being incubated withchromium labeled BT474M1 cells for 4 hours. Shown is percent lysis oftarget cells by chromium release at varying effector (activated NKcells):target (BT474M1) cell ratios cultured with media alone (●),anti-CD137 (▾), rituximab (▴), or rituximab and anti-CD137 (●)antibodies (p=0.006).

FIG. 14A-14B. Anti-CD137 agonistic mAb enhances anti-breast canceractivity of trastuzumab in-vivo. Nu/nu nude mice were inoculated with5×10⁶ BT474M1 breast tumor cells, subcutaneously, on the abdomen 1 dayafter subcutaneous injection of 0.72 mg/60 day release beta-estradiolpellet. (FIG. 14A-14B) Post-tumor inoculation, mice then received eitherRat IgG control on day 3 (●), trastuzumab antibody on day 3 (▪),anti-CD137 antibody on day 4 (♦), or trastuzumab on day 3 and anti-CD137antibody on day 4 (▴) with each treatment repeated weekly for a total ofthree weeks. Mice (10 per group) were then monitored for tumor growth(FIG. 14A, *p<0.001) and overall survival (FIG. 14B, **p=0.003).

FIG. 15A-15C. Anti-CD137 agonistic mAb enhances anti-breast canceractivity of trastuzumab in-vivo while retaining HER2 specificity. Nu/nunude mice were inoculated with 5×10⁶ MCF7 breast tumor cells,subcutaneously, on the left flank, and 5×10⁶ HER18 breast tumor cells,subcutaneously, on the right flank 1 day after subcutaneous injection of0.72 mg/60 day release beta-estradiol pellet. FIG. 15A-15C) Post-tumorinoculation, mice then received either trastuzumab on day 3, ortrastuzumab on day 3 and anti-CD137 antibody on day 4 with eachtreatment repeated weekly for a total of three weeks. FIG. 15A) Tumormodel. FIG. 15B) Representative mice (3 of 10 per group) were thenmonitored for tumor growth. FIG. 15C) Tumor growth by treatment groupand tumor type including MCF7 on left flank (∘) and HER18 on the rightflank (□) of mice treated with trastuzumab, and MCF7 on the left flank(●) and HER18 on the right flank (▪) of mice treated with trastuzumaband anti-CD137 mAbs (*p<0.001).

FIG. 16. Anti-CD137 agonistic mAb enhances anti-breast cancer activityof trastuzumab in-vivo against HER2⁺ primary breast tumor. SCID micewere inoculated with 1×10⁶ HER2⁺ primary breast tumor cells byintramammary injection 24 hours after 200 cGy total body irradiation(TBI). On day 40 mice were randomized to one of four groups (5 mice pergroup) including IgG control with treatment on day 40 (●), trastuzumabon day 40 (▪), anti-CD137 mAb on day 41 (♦), or trastuzumab on day 40and anti-CD137 mAb on day 41 (▴). Treatment was repeated weekly in eachgroup for a total of three treatments. Mice were monitored for tumorgrowth (*p=0.016).

FIG. 17A-17B. Cetuximab induces CD137 upregulation on human NK cellsfollowing incubation with EGFR-positive tumor cells. Peripheral bloodfrom three healthy donors was analyzed for CD137 expression on CD3⁻CD56⁺NK cells after 24 hour culture with head and neck cancer cell lines andmedia alone, rituximab or cetuximab. (FIG. 17A) shows EGFR surfaceexpression on head and neck cancer cell lines (103, SCC4, PC1, SCC6).Histograms were colored according to the log 10-fold increase in MFI ofbreast cancer cell lines relative to isotype. (FIG. 17B) shows CD137expression from three healthy donors cultured on NK cells CD3⁻CD56⁺after 24 hour culture with variably expressing EGFR head and neck cancercell lines (SCC6, PC1, and SCC4).

FIG. 18A-18C. Anti-CD137 agonistic mAb increases cetuximab-mediated NKcell cytotoxicity on tumor cells as assayed by cell viability.Preactivated NK cells were purified before being incubated with SCC6,PC1, and SCC4 for 24 hours. (FIG. 18A-18C) shows percent of apoptotictarget cells by annexin and 7AAD viability staining after culture withtumor alone or tumor and NK cells with media, cetuximab, anti-CD137,cetuximab and anti-CD137 antibodies (FIG. 18A, SCC6, *p=0.002; FIG. 18B,PC1, **p=0.011; FIG. 18C, SCC4, ***p=0.001).

FIG. 19. Anti-CD137 agonistic mAb increases cetuximab-mediated NK cellcytotoxicity on tumor cells as assayed by chromium release. NK cellcytotoxicity on SCC6 tumor cells was analyzed in chromium release assay.Fresh and preactivated NK cells were purified before being incubatedwith chromium labeled SCC6 cells for 5 hours. Shown is percent lysis oftarget cells by chromium release at varying effector (fresh NKcells):target (SCC6) cell ratios cultured with media alone (●),anti-CD137 (▾), cetuximab (▴), or cetuximab and anti-CD137 (▪)antibodies (**p=0.003) or preactivated NK cells as effectors with SCC6targets and cetuximab and anti-CD137 (♦) antibodies (*p=0.002).

FIG. 20. Anti-CD137 agonistic mAb enhances anti-head and neck canceractivity of cetuximab in-vivo. Nu/nu nude mice were inoculated with3×10⁶ SCC6 head and neck tumor cells, subcutaneously, on the abdomen.Post-tumor inoculation, mice then received either rat IgG control on day21 (●), cetuximab on day 21 (▪), anti-CD137 antibody on day 22 (▴), orcetuximab on day 21 and anti-CD137 antibody on day 22 (▴) with eachtreatment repeated weekly for a total of three treatments. Mice (10 pergroup) were then monitored for tumor growth (A, *p<0.001).

FIG. 21A-21B—Circulating NK cells upregulate CD137 following cetuximabinfusion in patients with head and neck cancer. Fresh peripheral bloodfrom patient with head and neck cancer was analyzed for CD137 expressionon CD3⁻CD56⁺ NK cells. (FIG. 21A) shows the phase 0, biomarker, trialschema (NCT01114256). (FIG. 21B) shows the percentage of CD137+ cellsamong fresh, peripheral blood CD3− CD56+ NK cells prior to and followingcetuximab infusion.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Methods are provided to enhance the anti-tumor effect of monoclonalantibodies directed against tumor antigens. In the methods of theinvention, ADCC function is augmented and target cell killing isenhanced by sequential administration of a combination of antibodies.The combination of agents and sequential administration is shown toprovide for synergistic effects, relative to the administration of thesingle agents. Administration of a tumor-directed antibody up-regulatesthe expression of inducible costimulatory molecules such as CD137, OX40,GITR, CD30 or ICOS on NK cells which are innate immune effector cellscritical for ADCC. Subsequently, a second agonistic antibody isadministered to target the induced costimulatory molecules (includingbut not limited to anti-CD137, -OX40, -GITR, -CD30 or -ICOS). In someembodiments, expression of the aforementioned costimulatory moleculesfollowing administration of the tumor-directed antibody is evaluated todetermine the optimal time for dosing the second agent. Alternatively atiming period is determined empirically, and generally applied. Becausethe second antibody targets costimulatory molecules which have beeninducibly expressed on NK cells by the tumor-directed antibody, thismethods allows specific stimulation of NK cells that are implicated inADCC-mediated killing of the tumor cells, while sparing other NK cells,thereby limiting potential non specific side effects.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Methods recited herein may be carried out in any order of the recitedevents which is logically possible, as well as the recited order ofevents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

Inducible costimulatory molecule. As used herein, an induciblecostimulatory molecule is a polypeptide expressed on immune cells,including without limitation natural killer (NK) cells, which expressionis induced or significantly upregulated during activation of NK cells.Activation of the costimulatory molecule enhanced the effector cellfunction, for example increasing ADCC mediated by the activated NKcells. Such inducible costimulatory molecules are known to those ofskill in the art, and include, without limitation, CD137, OX40, GITR,CD30, ICOS, etc. Agonists of such molecules, including antibodies thatbind to and activate the costimulatory molecule, are of interest for themethods of the invention. Many such costimulatory molecules are membersof the tumor necrosis factor receptor family (TNFR). TNFR-relatedmolecules do not have any known enzymatic activity and depend on therecruitment of cytoplasmic proteins for the activation of downstreamsignaling pathways.

CD137. CD137, which may also be referred to as Ly63, ILA or 4-1BB is amember of the tumor necrosis factor (TNF) receptor family. Members ofthis receptor family and their structurally related ligands areimportant regulators of a wide variety of physiologic processes and playan important role in the regulation of immune responses. CD137 isexpressed by activated NK cells, T and B lymphocytes andmonocytes/macrophages. The gene encodes a 255-amino acid protein with 3cysteine-rich motifs in the extracellular domain (characteristic of thisreceptor family), a transmembrane region, and a short N-terminalcytoplasmic portion containing potential phosphorylation sites.Expression in primary cells is strictly activation dependent. The ligandfor the receptor is TNFSF9. Human CD137 is reported to bind only to itsligand. Agonists include the native ligand (TNFSF9), aptamers (seeMcNamara et al. (2008) J. Clin. Invest. 118: 376-386), and antibodies.

CD134. OX40 (CD134) and its binding partner, OX40L (CD252), are membersof the tumor necrosis factor receptor/tumor necrosis factor superfamilyand are expressed on activated T cells as well as on a number of otherlymphoid and non-lymphoid cells. OX40 and OX40L regulate cytokineproduction from T cells, antigen-presenting cells, natural killer cells,and natural killer T cells, and modulate cytokine receptor signaling.

GITR. Glucocorticoid-Induced TNFR-Related (GITR) protein belongs totumor necrosis factor receptor/tumor necrosis factor superfamily andstimulates both the acquired and innate immunity. It is expressed inseveral cells and tissues, including T and Natural Killer (NK) cells andis activated by its ligand, GITRL, mainly expressed on antigenpresenting cells and endothelial cells. GITR/GITRL system participatesin the development of autoimmune/inflammatory responses and potentiatesresponse to infection and tumors by mechanisms including NK-cellco-activation.

CD30. The transmembrane receptor CD30 (TNFRSF8) and its ligand CD30L(CD153, TNFSF8) are members of the tumor necrosis factor (TNF)superfamily and display restricted expression in subpopulations ofactivated immune cells. CD30 is a type I transmembrane glycoprotein ofthe TNF receptor superfamily. The ligand for CD30 is CD30L (CD153). Thebinding of CD30 to CD30L mediates pleiotropic effects including cellproliferation, activation, differentiation, and apoptotic cell death.

Inducible costimulator (ICOS). ICOS is a member of the CD28 family. ICOSexpression, may be readily detectable resting, but it upregulated uponactivation. ICOS and ICOS-L appear to be a monogamous pair. ICOScostimulation enhances effector functions.

“Inducible costimulatory molecule agonist” includes the native ligands,as described above, aptamers, antibodies specific for an induciblecostimulatory molecule that activate the receptor, and derivatives,variants, and biologically active fragments of antibodies thatselectively bind to an inducible costimulatory molecule. A “variant”polypeptide means a biologically active polypeptide as defined belowhaving less than 100% sequence identity with a native sequencepolypeptide. Such variants include polypeptides wherein one or moreamino acid residues are added at the N- or C-terminus of, or within, thenative sequence; from about one to forty amino acid residues aredeleted, and optionally substituted by one or more amino acid residues;and derivatives of the above polypeptides, wherein an amino acid residuehas been covalently modified so that the resulting product has anon-naturally occurring amino acid. Ordinarily, a biologically activevariant will have an amino acid sequence having at least about 90% aminoacid sequence identity with a native sequence polypeptide, preferably atleast about 95%, more preferably at least about 99%. The variantpolypeptides can be naturally or non-naturally glycosylated, i.e., thepolypeptide has a glycosylation pattern that differs from theglycosylation pattern found in the corresponding naturally occurringprotein.

Fragments of the ligand or antibodies specific for an induciblecostimulatory molecule, particularly biologically active fragmentsand/or fragments corresponding to functional domains, are of interest.Fragments of interest will typically be at least about 10 aa to at leastabout 15 aa in length, usually at least about 50 aa in length, but willusually not exceed about 200 aa in length, where the fragment will havea contiguous stretch of amino acids that is identical to the polypeptidefrom which it is derived. A fragment “at least 20 aa in length,” forexample, is intended to include 20 or more contiguous amino acids from,for example, an antibody specific for CD137, or from TNFSF9. In thiscontext “about” includes the particularly recited value or a valuelarger or smaller by several (5, 4, 3, 2, or 1) amino acids. The proteinvariants described herein are encoded by polynucleotides that are withinthe scope of the invention. The genetic code can be used to select theappropriate codons to construct the corresponding variants. Thepolynucleotides may be used to produce polypeptides, and thesepolypeptides may be used to produce antibodies by known methods. A“fusion” polypeptide is a polypeptide comprising a polypeptide orportion (e.g., one or more domains) thereof fused or bonded toheterologous polypeptide.

In some embodiments, the inducible costimulatory molecule agonist is anantibody. The term “antibody” or “antibody moiety” is intended toinclude any polypeptide chain-containing molecular structure with aspecific shape that fits to and recognizes an epitope, where one or morenon-covalent binding interactions stabilize the complex between themolecular structure and the epitope. Antibodies utilized in the presentinvention may be polyclonal antibodies, although monoclonal antibodiesare preferred because they may be reproduced by cell culture orrecombinantly, and can be modified to reduce their antigenicity.

Polyclonal antibodies can be raised by a standard protocol by injectinga production animal with an antigenic composition. See, e.g., Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,1988. When utilizing an entire protein, or a larger section of theprotein, antibodies may be raised by immunizing the production animalwith the protein and a suitable adjuvant (e.g., Freund's, Freund'scomplete, oil-in-water emulsions, etc.) When a smaller peptide isutilized, it is advantageous to conjugate the peptide with a largermolecule to make an immunostimulatory conjugate. Commonly utilizedconjugate proteins that are commercially available for such use includebovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). In orderto raise antibodies to particular epitopes, peptides derived from thefull sequence may be utilized. Alternatively, in order to generateantibodies to relatively short peptide portions of the protein target, asuperior immune response may be elicited if the polypeptide is joined toa carrier protein, such as ovalbumin, BSA or KLH. Alternatively, formonoclonal antibodies, hybridomas may be formed by isolating thestimulated immune cells, such as those from the spleen of the inoculatedanimal. These cells are then fused to immortalized cells, such asmyeloma cells or transformed cells, which are capable of replicatingindefinitely in cell culture, thereby producing an immortal,immunoglobulin-secreting cell line. In addition, the antibodies orantigen binding fragments may be produced by genetic engineering.Humanized, chimeric, or xenogeneic human antibodies, which produce lessof an immune response when administered to humans, are preferred for usein the present invention.

In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)₂, or other fragments) are useful as antibodymoieties in the present invention. Such antibody fragments may begenerated from whole immunoglobulins by ricin, pepsin, papain, or otherprotease cleavage. “Fragment,” or minimal immunoglobulins may bedesigned utilizing recombinant immunoglobulin techniques. For instance“Fv” immunoglobulins for use in the present invention may be produced bylinking a variable light chain region to a variable heavy chain regionvia a peptide linker (e.g., poly-glycine or another sequence which doesnot form an alpha helix or beta sheet motif).

By “enhancing efficacy” is meant an increase in ADCC-mediated apoptosisof tumor cells compared to level of apoptosis observed with a singleagent, e.g. a monoclonal antibody specific for a tumor cell. Bysynergistic, it is meant that a combination of agents provides for aneffect greater than a single agent, which effect may be greater than theadditive effect of the combined agents.

Tumor directed antibodies. A number of antibodies are currently inclinical use for the treatment of cancer, and others are in varyingstages of clinical development. Antibodies of interest for the methodsof the invention act through ADCC, and are typically selective for tumorcells, although one of skill in the art will recognize that someclinically useful antibodies do act on non-tumor cells, e.g. CD20.

There are a number of antigens and corresponding monoclonal antibodiesfor the treatment of B cell malignancies. One popular target antigen isCD20, which is found on B cell malignancies. Rituximab is a chimericunconjugated monoclonal antibody directed at the CD20 antigen. CD20 hasan important functional role in B cell activation, proliferation, anddifferentiation. The CD52 antigen is targeted by the monoclonal antibodyalemtuzumab, which is indicated for treatment of chronic lymphocyticleukemia. CD22 is targeted by a number of antibodies, and has recentlydemonstrated efficacy combined with toxin in chemotherapy-resistanthairy cell leukemia. Two new monoclonal antibodies targeting CD20,tositumomab and ibritumomab, have been submitted to the Food and DrugAdministration (FDA). These antibodies are conjugated withradioisotopes.

Monoclonal antibodies useful in the methods of the invention, which havebeen used in solid tumors, include without limitation edrecolomab andtrastuzumab (herceptin). Edrecolomab targets the 17-1A antigen seen incolon and rectal cancer, and has been approved for use in Europe forthese indications. Its antitumor effects are mediated through ADCC, CDC,and the induction of an anti-idiotypic network. Trastuzumab targets theHER-2/neu antigen. This antigen is seen on 25% to 35% of breast cancers.Trastuzumab is thought to work in a variety of ways: downregulation ofHER-2 receptor expression, inhibition of proliferation of human tumorcells that overexpress HER-2 protein, enhancing immune recruitment andADCC against tumor cells that overexpress HER-2 protein, anddownregulation of angiogenesis factors.

Alemtuzumab (Campath) is used in the treatment of chronic lymphocyticleukemia; colon cancer and lung cancer; Gemtuzumab (Mylotarg) finds usein the treatment of acute myelogenous leukemia; Ibritumomab (Zevalin)finds use in the treatment of non-Hodgkin's lymphoma; Panitumumab(Vectibix) finds use in the treatment of colon cancer.

Cetuximab (Erbitux) is also of interest for use in the methods of theinvention. The antibody binds to the EGF receptor (EGFR), and has beenused in the treatment of solid tumors including colon cancer andsquamous cell carcinoma of the head and neck (SCCHN).

The term “biological sample” encompasses a variety of sample typesobtained from an organism and can be used in a diagnostic or monitoringassay. The term encompasses blood and other liquid samples of biologicalorigin, solid tissue samples, such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. The termencompasses samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components. The term encompasses a clinicalsample, and also includes cells in cell culture, cell supernatants, celllysates, serum, plasma, biological fluids, and tissue samples.

The terms “cancer”, “neoplasm”, “tumor”, and “carcinoma”, are usedinterchangeably herein to refer to cells which exhibit relativelyautonomous growth, so that they exhibit an aberrant growth phenotypecharacterized by a significant loss of control of cell proliferation. Ingeneral, cells of interest for detection or treatment in the presentapplication include precancerous (e.g., benign), malignant,pre-metastatic, metastatic, and non-metastatic cells. Detection ofcancerous cells is of particular interest. The term “normal” as used inthe context of “normal cell,” is meant to refer to a cell of anuntransformed phenotype or exhibiting a morphology of a non-transformedcell of the tissue type being examined. “Cancerous phenotype” generallyrefers to any of a variety of biological phenomena that arecharacteristic of a cancerous cell, which phenomena can vary with thetype of cancer. The cancerous phenotype is generally identified byabnormalities in, for example, cell growth or proliferation (e.g.,uncontrolled growth or proliferation), regulation of the cell cycle,cell mobility, cell-cell interaction, or metastasis, etc. Cancers ofinterest include, without limitation, hematopoietic cancers includingleukemias, lymphomas (Hodgkins and non-Hodgkins), myelomas andmyeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomasof solid tissue, squamous cell carcinomas of the mouth, throat, larynx,and lung, liver cancer, genitourinary cancers such as cervical, bladdercancer and renal cell carcinomas, head and neck cancers, gastrointestinal track cancers and nervous system cancers, benign lesions suchas papillomas, and the like.

The phrase “solid tumor” as used herein refers to an abnormal mass oftissue that usually does not contain cysts or liquid areas. Solid tumorsmay be benign or malignant. Different types of solid tumors are namedfor the type of cells that form them. Examples of solid tumors aresarcomas, carcinomas, lymphomas etc.

Monoclonal antibodies directed against a specific cancer epitope, orcombination of epitopes allows the targeting and/or depletion of cancercell populations expressing the marker. Various techniques can beutilized using monoclonal antibodies to screen for cellular populationsexpressing the marker(s), and include magnetic separation usingantibody-coated magnetic beads, “panning” with antibody attached to asolid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No.5,985,660; and Morrison et al. Cell, 96:737-49 (1999)). These techniquesallow for the screening of particular populations of cells; inimmunohistochemistry of biopsy samples; in detecting the presence ofmarkers shed by cancer cells into the blood and other biologic fluids,and the like.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing the diseaseor symptom from occurring in a subject which may be predisposed to thedisease or symptom but has not yet been diagnosed as having it; (b)inhibiting the disease symptom, i.e., arresting its development; or (c)relieving the disease symptom, i.e., causing regression of the diseaseor symptom.

The terms “recipient”, “individual”, “subject”, “host”, and “patient”,used interchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans.

“Therapeutic target” refers to molecules expressed by the tumor cellsand/or non tumor (immune) cells that can be targeted to induce orenhance antitumor activity.

Methods

Methods are provided to enhance the efficacy of cell killing induced bythe administration of antibodies directed against tumor cells. Aneffective dose of a primary tumor-directed antibody is administered to apatient, which induces the upregulation of inducible costimulatorymolecules such as CD137, OX40, GITR, CD30 or ICOS on NK cells, which areinnate immune effector cells critical for ADCC. Subsequently, aneffective dose of a second agonistic antibody against one of thesemolecules, including but not limited to CD137, OX40, GITR, CD30 or ICOS)is administered to said individual, where the second antibody issufficient to enhance ADCC killing of tumor cells targeted by the firstantibody. Because the second antibody targets costimulatory moleculesthat have been inducibly expressed on NK cells by the tumor-directedantibody, this methods allows specific stimulation of NK cells that areimplicated in ADCC-mediated killing of the tumor cells, while sparingother NK cells, thereby limiting potential non-specific side effects.

In some embodiments the level of costimulatory molecules (including butnot limited to CD137, OX40, GITR, CD30 or ICO) induced by thetumor-directed antibody is determined in a patient sample, usually apatient blood sample or cellular fraction thereof. As a baseline, thelevel of costimulatory molecules may be determined in a sample prior toadministering the tumor-directed antibody, and the increase inexpression following administration of the tumor-directed antibodydetermined.

In some embodiments of the invention, an effective dose of atumor-selective antibody is administered to a patient, following whichsufficient time is elapsed for an upregulation of CD137 expression onimmune system cells, particularly NK cells. The sufficient time isusually at least about 12 hours, more usually at least about 18 hours,and usually at least about 24 hours, and may be at least about 2 days,at least about 3 days, and not more than about 5 days, usually not morethan about 4 days. Following upregulation of CD137, an effective dose ofa CD137 agonist is administered to said individual, where the agonist issufficient to enhance ADCC killing of tumor cells targeted by the firstantibody. In some embodiments the level of CD137 is determined in apatient sample, usually a patient blood sample or cellular fractionthereof. As a baseline, the level of CD137 may be determined in a sampleprior to administering the tumor-selective antibody, and the increase inexpression following administration of the tumor-selective antibodydetermined. A desirable increase in expression of CD137 on NK cells isat least about 1.5-fold, at least about 2-fold, or higher. An increasein expression when measured in overall blood cells may be lower due tothe number of contaminating non-responsive cells.

“Reducing growth of cancer cells” includes, but is not limited to,reducing proliferation of cancer cells, and increasing apoptosis oftumor cells. Whether a reduction in cancer cell growth has been achievedcan be readily determined using any known assay, including, but notlimited to, [³H]-thymidine incorporation; counting cell number over aperiod of time; detecting and/or measuring a marker associated with thecancer of interest, etc.

Whether a substance, or a specific amount of the substance, is effectivein treating cancer can be assessed using any of a variety of knowndiagnostic assays for cancer, including, but not limited to biopsy,contrast radiographic studies, CAT scan, and detection of a tumor markerassociated with cancer in the blood of the individual. The substance canbe administered systemically or locally, usually systemically.

Formulations

Therapeutic formulations comprising one or more antibodies utilized inthe methods of the invention may be prepared for storage by mixing theantibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Theantibody composition will be formulated, dosed, and administered in afashion consistent with good medical practice. Factors for considerationin this context include the particular cancer being treated, theclinical condition of the individual patient, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the antibody to be administeredwill be governed by such considerations.

The therapeutic dose may be at least about 0.01 μg/kg body weight, atleast about 0.05 μg/kg body weight; at least about 0.1 μg/kg bodyweight, at least about 0.5 μg/kg body weight, at least about 1 μg/kgbody weight, at least about 2.5 μg/kg body weight, at least about 5μg/kg body weight, and not more than about 100 μg/kg body weight. Itwill be understood by one of skill in the art that such guidelines willbe adjusted for the molecular weight of the active agent, e.g. in theuse of antibody fragments, or in the use of antibody conjugates. Thedosage may also be varied for route of administration.

Acceptable carriers, excipients, or stabilizers are non-toxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Formulations to be used for in vivo administration must be sterile. Thisis readily accomplished by filtration through sterile filtrationmembranes.

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for treating the condition and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The active agent in the composition is one or more antibodiesas described above. The label on, or associated with, the containerindicates that the composition is used for treating the condition ofchoice. The article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

Antibodies and agonists suitable for use in the methods of the inventionmay be provided in a kit form, for example presented in a pack ordispenser device containing one or more unit dosage forms containing theactive ingredients. Such a pack or device may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.Compositions comprising agents useful in the methods of the inventionmay be formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition. Suitable conditions indicated on the labelmay include treatment of cancer. Kits may also comprise a unit suitablefor measuring expression of an inducible costimulatory molecule on NKcells, e.g. including detectable labeled reagent that specifically bindsto an inducible costimulatory molecule, and references for expression,and the like as known in the art.

EXPERIMENTAL

The paradigm of cancer treatment prior to a decade ago was limited toconventional cytotoxic chemotherapy with the assumption of increasedsensitivity to agents such as cell cycle inhibitors or DNA damagingagents by rapidly proliferating tumor cells. This paradigm changed in1997 with the Food and Drug Administration approval of rituximab, thefirst therapeutic monoclonal antibody, which targets the surface markerCD20 for the treatment of non-Hodgkin lymphoma. Monoclonal antibodieshave unique advantages over conventional cytotoxicchemotherapy—well-tolerated with less systemic toxicity, selectivity totumor with minimal off-target effects, and ability to target slowlyproliferating cells not undergoing frequent mitosis.

Following the approval of rituximab, trastuzumab, a humanized IgG1antibody against HER2 (human epidermal growth factor receptor 2), wasapproved in 1998 for HER2⁺ breast cancer. In 2004 cetuximab, a chimericIgG1 antibody targeting the EGFR (epidermal growth factor receptor), wasapproved for colorectal cancer. In 2006 cetuximab was also approved fortreatment of head and neck cancers. As evidenced by the recentidentification of HER2+ gastric and esophageal cancer, cancer therapiesare now focused on discovery of both novel and known targets to whichnew or current antibodies can be aimed.

In contrast to conventional chemotherapy, monoclonal antibodiesdemonstrate minimal direct cytotoxicity. Though evidence supportsmultiple mechanisms of antibody function, a major mechanism of antitumoractivity is through antibody dependent cell-mediated cytotoxicity(ADCC). Cytotoxicity occurring by ADCC is mediated by natural killer(NK) cells or macrophage/monocytes bearing an Fc receptor which binds tothe antibody-targeted tumor cell. NK cell Fc receptor binding toantibody activates the NK cell resulting in release of cytokines andcytotoxic granules which trigger apoptosis in the antibody-targetedtumor cell. Increased NK cell function augments ADCC and results inimproved antitumor activity.

The present invention identifies costimulatory molecules that areinducibly expressed on NK cells, which are innate effector cellscritical for ADCC following recognition of antibody-coated tumor cells.Following up-regulation by the tumor directed antibody, thesecostimulatory molecules (including but not limited to CD137, OX40, GITR,CD30 or ICOS) can subsequently be targeted with agonistic antibodies toenhance ADCC mediated by these effector cells. Given the increasingnumber of tumor target-specific antibodies, this has therapeuticimplications for any cancer type directly targeted by a monoclonalantibody.

The clinical benefit of agonistic antibodies that enhance NK cellfunction is limited only by the number of targets expressed by tumorcells and target-specific antibodies. Given the impact of rituximab,trastuzumab and cetuximab over the prior decade, target and antibodydiscovery efforts are likely to continue to be an area of activeresearch. Though application of agonistic NK cell antibodies will extendbeyond non-Hodgkin lymphoma, breast cancer, colorectal and head and neckcancers, these four demonstrate the magnitude of clinical benefit.

Antibody coated tumor cells trigger the activation of NK cells, whosekilling activity can then be stimulated by a second activating antibodyagainst CD137. The addition of agonistic anti-CD137 antibody is ageneral approach to enhance the therapeutic effect of any anti-tumorantibody.

Example 1

Increased NK cell expression of CD137 occurs following NK cell exposureto CD20⁺ lymphoma coated with rituximab. Targeting CD137 with anagonistic antibody to enhance antitumor ADCC is dependent upon itsincreased surface expression on NK cells following their exposure toantibody coated tumor cells. Because the second antibody targets acostimulatory molecule (CD137) which is inducibly expressed on NK cellsby the tumor-directed antibody (rituximab, trastuzumab), this methodsallows specific stimulation of NK cells which are implicated inADCC-mediated killing of the tumor cells (lymphoma, breast cancer),while sparing other NK cells, thereby limiting potential non specificside effects.

We isolated peripheral blood mononuclear cells (PBMCs) from patientswith circulating CD20⁺ tumor cells due to chronic lymphocytic leukemia(CLL), marginal zone lymphoma (MZL), and CD20⁺ acute lymphoblasticleukemia (ALL). PBMCs were analyzed by flow cytometry after culture withtrastuzumab or rituximab. Percent CD137⁺ NK cells increased from 1-2% atbaseline to 22-43% with concurrent downregulation of CD16 (the Fcreceptor) following culture with rituximab. This appeared antibodydependent since no activation occurred following culture withtrastuzumab. As shown in FIGS. 1A-1C, Rituximab induces CD137upregulation on human NK cells following incubation with CD20-positivetumor B cells. Peripheral blood from three healthy donors was analyzedfor CD137 expression on CD3⁻CD56⁺ NK cells after 24 hour culture withlymphoma cell lines and trastuzumab or rituximab.

Anti-CD137 agonistic mAb increases rituximab-mediated NK cellcytotoxicity on tumor cells, as shown in FIGS. 2A-2F. NK cells isolatedand purified from the peripheral blood of healthy donors were analyzedfor degranulation by CD107a mobilization after 24 hour culture with fiveconditions: media alone; CD20-positive lymphoma cell line (Raji, Ramos,or DHL-4); tumor and rituximab; tumor and anti-CD137 antibody; or tumor,rituximab, and anti-CD137 agonistic antibody. Shown in FIGS. 3A-3D,there is also an enhancement of anti-lymphoma activity with anti-CD137agonistic mAb.

Anti-CD20 and anti-CD137 mAbs combination activity requires appropriatesequence of mAb administration, as shown in FIGS. 4A-4B, and isdependent on NK cells and macrophages. Shown in FIGS. 5A-5D, peripheralblood cell subsets from lymphoma-bearing C57BL/6 mice 4 days post-tumorinoculation treated on day 3 with either IgG control or anti-CD20antibody were analyzed for CD137 expression on CD3-NK1.1⁺ NK cells (NK),F4/80⁺ macrophages (Mφ), CD3⁺CD8⁺ T cells (CD8), and CD3⁺CD4⁺ T cells(CD4); Tumor-infiltrating lymphocytes from lymphoma-bearing C57BL/6 mice7 days post-tumor inoculation treated on day 3 with either IgG controlor anti-CD20 antibody were analyzed for CD137 expression on CD3-NK1.1⁺NK cells (NK), F4/80⁺ macrophages (Mφ), CD3⁺CD8⁺ T cells (CD8), andCD3⁺CD4⁺ T cells.

The synergy of anti-CD20Ab and anti-CD137Ab therapy observed in thesyngeneic murine model is validated by testing the activity ofanti-CD137Ab with rituximab, trastuzumab, and cetuximab in xenograftathymic, nude mouse models. This model has been previously used forpreclinical testing of rituximab, trastuzumab, and cetuximab. In thelymphoma model, Balb/c nude nu/nu mice are inoculated on day 0 with3×10⁶ Raji cells transfected with Firefly Luciferase, and treated withrituximab (10 μg/g weight, IP) on day 3 followed by 150 μg of ratanti-mouse anti-CD137Ab IP on day 4. Blood is collected on day 3 priorto rituximab treatment, on day 4 prior to anti-CD137Ab treatment, and onday 5 to assay NK cell expression of CD137, CD69, and CD16.Bioluminescent imaging is performed following IP luciferin injection(200 μL) on day 3 prior to Ab therapy and repeated weekly. The effect ofCD137 with rituximab is shown in a disseminated human lymphomaxenotransplant model, shown in FIGS. 6A-6C.

Rituximab-coated, autologous lymphoma cells induce CD137 upregulation onNK cells from human patients with B cell malignancies, shown in FIGS.7A-7C. Peripheral blood from patients with B cell malignancies andcirculating tumor cells (CTC) were analyzed for CD137 expression onCD3⁻CD56⁺ NK cells after 24 hour culture with media alone, trastuzumab,or rituximab. The kinetics of CD137 induction and temporal expression onNK cells following preactivation are analyzed in FIGS. 8A-8C.

Tumor growth was reduced by approximately 50% with either anti-CD20Ab oranti-CD137Ab monotherapy. However, all mice treated with anti-CD20Abdied before 60 days, and only 50% of mice treated with anti-CD137Ab werealive 100 days post-tumor inoculation. Treatment with anti-CD20Ab on day3 followed by anti-CD137Ab on day 4 resulted in complete regression oftumor and survival at 100 days in 90% of mice. To determine if theobserved synergy is dependent on NK cell function, NK cells weredepleted with anti-asialo-GM1 on day −1, 0, and every 5 days thereaftertill 20 days at which point a clear separation of treatment groups wasobserved. NK cell depletion with anti-asialo-GM1 abrogated the benefitof combination therapy.

Anti-CD137 agonistic mAb increases cytokine release andrituximab-mediated cytotoxicity of pre-activated NK cells, shown inFIGS. 9A-9B. To evaluate NK cell interferon-γ secretion purified NKcells were isolated from healthy PBMCs and cultured for 24 hourstogether with rituximab (10 μg/mL) and irradiated (5,000 rads) lymphomatumor cells (Raji) at a ratio of 1:1. After 24 hours, NK cells wereisolated and assessed for purity. Preactivated, purified NK cells werethen cultured for 4 hours in media alone, or with anti-CD137 mAb alone,rituximab alone, or rituximab plus anti-CD137 mAbs and supernatant washarvested and analyzed by ELISA for interferon-γ. NK cell cytotoxicityon Raji tumor cells was analyzed in chromium release assay with andwithout prior NK cell preactivation. Anti-CD137 agonistic mAb alsoincreases rituximab-mediated NK cell degranulation, shown in FIG. 10.

Example 2

Increased NK cell expression of CD137 occurs following NK cell exposureto HER2′ breast cancer coated with trastuzumab. We then determined ifexpression of CD137 is similarly increased on NK cells following theirexposure to breast cancer coated with trastuzumab. We isolated NK cellsfrom blood of healthy donors and added them to breast cancer cell linesincluding MCF7 (a non-HER2 expressing breast cancer cell line) and SKBR3(a HER2 overexpressing breast cancer cell line) for 24 hours togetherwith trastuzumab or rituximab. The NK cells were then analyzed by flowcytometry for CD137 expression, shown in FIG. 11.

NK cell expression of CD137 was determined following co-culture withappropriate tumor cell lines coated with cetuximab (10 μg/mL), rituximab(10 μg/mL) or trastuzumab (10 μg/mL) as detailed above for breast cancercell lines. Flow cytometry for CD3 and CD56 was performed to evaluatepurity of the NK cell isolation. Additional markers of activationincluding CD69, CD107, and CD16 were included in the flow cytometrypanel. The colon cancer cell lines include HCT-8 (EGFR⁺) and SW620(EGFR), and the squamous head and neck cancer cell lines include TE3(EGFR⁺HER2⁻) and TE4 (EGFR⁻HER2⁺).

As shown in FIGS. 12A-12C, anti-CD137 agonistic mAb increasestrastuzumab-mediated NK cell cytotoxicity on tumor cells as assayed bycell viability. Preactivated NK cells were purified before beingincubated with MCF7, BT474M1, and HER18 for 18 hours, and apoptosisevaluated

The functional capacity of activated NK cells following theirstimulation with antibody and tumor cells was also determined bychromium release assays. To investigate activity against HER2⁺ breastcancer, NK cells isolated from normal blood were activated by co-culturewith SKBR3 (HER2⁺) and trastuzumab. These exposed NK cells were testedby flow cytometry for CD137 and CD69 expression to evaluate theiractivation. Activated NK cells are added to chromium labeled SKBR3target cells at ratios of 12.5:1, 25:1, 50:1, and 100:1 together withtrastuzumab (10 μg/mL), anti-CD137Ab (10 μg/mL), ortrastuzumab+anti-CD137Ab (both at 10 μg/mL). A similar activation andkilling assay is performed to investigate in-vitro functional activityagainst EGFR⁺ colon cancer with HCT-8 (EGFR⁺) cell line and cetuximab,and EGFR⁺ head and neck cancers with TE3 (EGFR⁺HER2⁻) cell line andcetuximab. Shown in FIG. 13, anti-CD137 agonistic mAb increasestrastuzumab-mediated NK cell cytotoxicity on tumor cells as assayed bychromium release.

Supporting evidence has been provided in a syngenic murine lymphomamodel, anti-CD137 agonistic mAb enhances anti-breast cancer activity oftrastuzumab in-vivo. As shown in FIGS. 14A-14B. Nu/nu nude mice wereinoculated with 5×10⁶ BT474M1 breast tumor cells, subcutaneously, on theabdomen 1 day after subcutaneous injection of 0.72 mg/60 day releasebeta-estradiol pellet. Post-tumor inoculation, mice then received eitherrat IgG control on day 3, trastuzumab antibody on day 3, anti-CD137antibody on day 4, or trastuzumab on day 3 and anti-CD137 antibody onday 4 with each treatment repeated weekly for a total of three weeks.Mice were then monitored for tumor growth and overall survival.

As shown in FIG. 15A-15C, anti-CD137 agonistic mAb enhances anti-breastcancer activity of trastuzumab in-vivo while retaining HER2 specificity.Nu/nu nude mice were inoculated with 5×10⁶ MCF7 breast tumor cells,subcutaneously, on the left flank, and 5×10⁶ HER18 breast tumor cells,subcutaneously, on the right flank 1 day after subcutaneous injection of0.72 mg/60 day release beta-estradiol pellet. Post-tumor inoculation,mice then received either trastuzumab on day 3, or trastuzumab on day 3and anti-CD137 antibody on day 4 with each treatment repeated weekly fora total of three weeks. Representative mice were then monitored fortumor growth

Anti-CD137 agonistic mAb enhances anti-breast cancer activity oftrastuzumab in-vivo against HER2⁺ primary breast tumor, as shown in FIG.16. SCID mice were inoculated with 1×10⁶ HER2⁺ primary breast tumorcells by intramammary injection 24 hours after 200 cGy total bodyirradiation (TBI). On day 40 mice were randomized to one of four groupsincluding IgG control with treatment on day 40, trastuzumab on day 40,anti-CD137 mAb on day 41, or trastuzumab on day 40 and anti-CD137 mAb onday 41. Treatment was repeated weekly in each group for a total of threetreatments. Mice were monitored for tumor growth.

Example 3

Cetuximab induces CD137 upregulation on human NK cells followingincubation with EGFR-positive tumor cells, as shown in FIGS. 17A-17B.Peripheral blood from three healthy donors was analyzed for CD137expression on CD3⁻CD56⁺ NK cells after 24 hour culture with head andneck cancer cell lines and media alone, rituximab or cetuximab, andshown to have increased expression.

As shown in FIGS. 18A-18C, anti-CD137 agonistic mAb increasescetuximab-mediated NK cell cytotoxicity on tumor cells as assayed bycell viability. Preactivated NK cells were purified before beingincubated with SCC6, PC1, and SCC4 for 24 hours. The percent ofapoptotic target cells was determined by annexin and 7AAD viabilitystaining after culture with tumor alone or tumor and NK cells withmedia, cetuximab, anti-CD137, cetuximab and anti-CD137 antibodies.

Anti-CD137 agonistic mAb increases cetuximab-mediated NK cellcytotoxicity on tumor cells as assayed by chromium release. NK cellcytotoxicity on SCC6 tumor cells was analyzed in chromium release assay.Fresh and preactivated NK cells were purified before being incubatedwith chromium labeled SCC6 cells for 5 hours. Shown in FIG. 19 ispercent lysis of target cells by chromium release at varying effector(fresh NK cells):target (SCC6) cell ratios.

Anti-CD137 agonistic mAb enhances anti-head and neck cancer activity ofcetuximab in-vivo. Nu/nu nude mice were inoculated with 3×10⁶ SCC6 headand neck tumor cells, subcutaneously, on the abdomen, shown in FIG. 20.Post-tumor inoculation, mice received either rat IgG control on day 21,cetuximab on day 21, anti-CD137 antibody on day 22, or cetuximab on day21 and anti-CD137 antibody on day 22 with each treatment repeated weeklyfor a total of three treatments. Mice were then monitored for tumorgrowth.

It was additionally shown that circulating NK cells upregulate CD137following cetuximab infusion in patients with head and neck cancer(FIGS. 21A-21B). Fresh peripheral blood from patient with head and neckcancer was analyzed for CD137 expression on CD3⁻CD56⁺ NK cells, andshown to be upregulated.

The demonstration that an activation specific target (CD137) appears onNK cells after their exposure to antibody coated tumor cells, and that asecond antibody against the CD137 target on these host cells hassynergistic antitumor activity in multiple tumor models is innovativeand of high impact to patient care. Agonistic anti-CD137 antibodies (forexample BMS-663513) are currently in early-phase clinical trials, and inpreclinical development by multiple major pharmaceutical companies.Approximately 300 patients have been treated in phase I and II trialswith the BMS antibody. Negligible single agent activity has beenobserved with response rates less than 10%.

Based on our in vitro data, without concurrent tumor-targeted antibodysuch as rituximab no significant increase in NK cell function isobserved. However, NK cell function is dramatically increased when bothantibodies are combined. The present invention is of high importance asit has the opportunity to demonstrate the clinical value of anti-CD137combination antibody therapy, despite minimal single agent activity insolid tumors. Therapy based on synergy through immunomodulation is aninnovative and paradigm changing approach to improving the survival ofpatients with many types of cancer.

More broadly, we have observed that a number of other costimulatorymolecules beside CD137, such as OX40 and GITR are up-regulated on NKcells following exposure to antibody-coated tumor cells. Similar toCD137, targeting of these inducible costimulatory molecules withagonistic antibodies are expected to stimulate and enhance NK cellfunction leading to increased ADCC.

What is claimed is:
 1. A method of treating cancer, the methodcomprising: administering to an individual a first antibody selectivefor a cancer cell antigen, and after a period of time sufficient toinduce or upregulate expression of an inducible costimulatory moleculeon NK cells; administering to said individual an agonist of saidinducible costimulatory molecule in a dose effective to enhance Nk cellantibody-dependent cellular cytotoxicity (ADCC); wherein the populationof tumor cells in said patient is decreased.
 2. The method of claim 1,wherein said inducible costimulatory molecule is one or more of CD137,OX40, GITR, CD30 and ICOS.
 3. The method of claim 1, wherein saidagonist is a monoclonal antibody.
 4. The method of claim 3, wherein thefirst antibody is specific for at least one of CD20, CD19, CD22, CD52,Her2, and epidermal growth factor receptor (ERFR).
 5. The method ofclaim 1, wherein the method provides a synergistic effect in decreasingthe population of tumor cells.
 6. The method of claim 1, furthercomprising the step of measuring expression of said induciblecostimulatory molecule on NK cells in said patient prior toadministering said first antibody and prior to administering saidagonist, wherein said agonist is administered after an increase inexpression of said inducible costimulatory molecule on NK cells.
 7. Themethod of claim 1, wherein the cancer is a lymphoma.
 8. The method ofclaim 1, wherein the cancer is a solid tumor.
 9. The method of claim 8,wherein the solid tumor is a breast carcinoma.
 10. The method of claim8, wherein the solid tumor is a colon cancer.
 11. The method of claim 8,wherein the solid tumor is a head and neck cancer.
 12. The method ofclaim 1, wherein the antibody selective for a cancer cell antigen isspecific for CD20.
 13. The method of claim 1, wherein the antibodyselective for a cancer cell antigen is specific for her-2.
 14. Themethod of claim 1, wherein the antibody selective for a cancer cellantigen is specific for epidermal growth factor receptor (ERFR).
 15. Themethod of claim 1, wherein the antibody selective for a cancer cellantigen is specific for CD52.
 16. The method of claim 1, wherein theantibody selective for a cancer cell antigen is specific for CD19. 17.The method of claim 1, wherein the antibody selective for a cancer cellantigen is specific for CD22.